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Ann Thorac Surg 1998;65:1680-1684
© 1998 The Society of Thoracic Surgeons

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Original articles: cardiovascular

Postischemic Function and Protein Kinase C Signal Transduction

^a Section of Thoracic Surgery, University of Michigan Medical Center, Ann Arbor, Michigan, USA

Accepted for publication January 31, 1998.

Address reprint requests to Dr Bolling, Section of Thoracic Surgery, The University of Michigan Hospitals, 1500 E Medical Center Dr, 2120D Taubman Center, Box 0344, Ann Arbor, MI 48109
e-mail: (sbolling{at}umich.edu)

	Abstract

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Background. The protective effects of myocardial preconditioning may occur by way of multiple mechanisms, with G-protein-mediated protein kinase C (PKC) translocation as a final common pathway. In this study we investigate the pharmacologic induction of preconditioning, by PKC translocation, using PKC agonists/antagonists to reveal its effects on contractile function after myocardial ischemia.

Methods. Langendorff-perfused rabbit hearts received: (1) control; (2) dimethyl sulfoxide (vehicle); (3) acetylcholine (0.55 mmol/L; PKC agonist); (4) 1,2-s,n-dioctanoylglycerol (DOG; 22 mmol/L; PKC agonist); (5) chelerythrine (0.8 mmol/L; PKC antagonist); or (6) DOG–chelerythrine followed by a 2-hour ischemic period, using modified St. Thomas cardioplegia and a 45-minute reperfusion period. The period of ischemia was chosen so as to allow for improvement by appropriate agonists. To observe metabolic changes, tissue nucleotides and nucleosides were measured. Membrane and cytosolic fractions of PKC were determined by an anti-PKC antibody directed against the PKC isozyme. Lactate levels and myocardial pH were measured.

Results. The PKC agonists DOG and acetylcholine showed the greatest recovery of developed pressure (68% ± 2%, 60% ± 9%, respectively). Although pH, lactate, and nucleotide levels were similar between groups at all times, myocyte PKC translocation demonstrated 25% of PKC isoforms on cell membrane sites during baseline, which shifted to 67% ± 17% with unprotected ischemia. DOG mimicked this shift with 58% ± 12% of PKC isoforms on membranes, which was also blocked by chelerythrine to 35% ± 7%.

Conclusions. These data demonstrate that PKC translocation results in improved postischemic function, not by alteration of energetics or metabolism, and deserves further investigation.

	Introduction

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Despite the use of cardioplegia to protect the heart during surgically induced ischemia, the ideal method of myocardial protection remains to be established. Myocardial injury can be reduced by cold cardioplegia but protection is not complete and poor functional recovery often results. In studies of infarct modification, infarct size reduction is achieved with brief episodes of ischemia before prolonged ischemia, a phenomenon known as myocardial preconditioning. Identification of the intracellular mechanisms that invoke the preconditioning phenomenon may provide important information to develop novel strategies for myocardial protection during prolonged periods of surgically induced cardiac arrest. There is evidence that preconditioning is induced by multiple signaling pathways, including adenosine A1 receptor, muscarinic M2 receptor, and ₁-adrenergic receptors, which have as a final common pathway G-protein modulation of protein kinase C (PKC) activity. The PKC translocation from cytosol to membrane surface has been hypothesized to lead to an adaptive change in viability and contractility.

The specific objective of this study was to demonstrate whether pharmacologic activation and translocation of PKC from cytosol to cell membrane is a pathway that may mimic ischemic preconditioning and correlates with improved functional recovery after global myocardial ischemia. The rabbit isolated heart infused with cardioplegia was used in an attempt to simulate the clinically relevant condition of surgically induced ischemia.

	Material and methods

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Animals
New Zealand white rabbits weighing 2.2 to 2.7 kg (either sex) were used in all studies. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research.

Isolated rabbit heart preparation
Rabbits were anesthetized with intravenous sodium pentobarbital (45 mg/kg) and heparin (700 U/kg). The heart was rapidly excised and immersed in ice-cold physiologic salt solution, pH 7.4, containing 118.0 mmol/L NaCl, 4.0 mmol/L KCl, 22.3 mmol/L NaHCO₃, 11.1 mmol/L glucose, 0.66 mmol/L KH₂PO₄, 1.23 mmol/L MgCl₂, and 2.38 mmol/L CaCl₂. The aorta was cannulated in the Langendorff mode and the heart was perfused with physiologic salt solution equilibrated with 95% O₂–5% CO₂ at 37°C and passed twice through filters with 3.0-nm pore size. Perfusion pressure was maintained at 90 mm Hg. An incision was made in the left atrium, and a fluid-filled latex balloon was passed through the mitral orifice and placed in the left ventricle. The balloon was connected to a pressure transducer for continuous measurement of left ventricular pressure and its first derivative. The caudal vena cave, the left and right cranial vena cave, and the azygous vein were ligated. The pulmonary artery was cannulated to enable timed collection measurements of coronary flow and the cannula was connected to an oxygen meter (Diamond Electro-Tech, Inc, Ann Arbor, MI) for continuous measurement of the oxygen partial pressure. The analog signals were continuously recorded on a pressurized ink chart recorder (model 2600S; Gould, Inc, Cleveland, OH) and digitized to an on-line computer (AST Premium/386; AST Research Inc, Irvine, CA). To characterize cardiac function, developed pressure is defined as peak systolic pressure minus end-diastolic pressure. Myocardial oxygen consumption (MVO₂) was calculated as where CF is coronary flow (mV · min^-1 · g^-1), (PaO₂ - PvO₂) is the difference in the partial pressure of oxygen (PO₂, mm Hg) between perfusate and coronary effluent flow, and c is the Bunsen solubility coefficient of O₂ in perfusate at 37°C (22.7 µL O₂ · atm^-1 · mL^-1 perfusate). The PO₂ of the perfusate was 665 mm Hg. Coronary flow was measured by performing timed collections of the pulmonary effluent flow with a graduated cylinder. Oxygen extraction (O₂ EXT) was calculated as After completing instrumentation and performing calibrations, left ventricular balloon volumes were varied over a range of values to construct modified left ventricular function curves. This volume was maintained the same during baseline and reperfusion conditions.

Experimental protocol
Baseline data was obtained after an equilibration period of approximately 30 minutes, with the hearts maintained at 37°C by passing saline at this temperature through the organ bath. During ischemia, however, the organ bath temperature was reduced to 34°C. After preperfusion with PKC agonists/antagonists, 60 mL of modified St. Thomas cardioplegic solution (4°C) was injected into the aorta at a rate of 1 mVs and the physiologic salt solution infusion stopped to begin the 2-hour ischemic period. Fifteen milliliters of modified St. Thomas cardioplegic solution (4°C) was injected every 30 minutes thereafter. Control hearts received oxygenated cardioplegia containing 109.0 mmol/L NaCl, 25.0 mmol/L KCl, 13.5 mmol/L NaHCO₃, 16.0 mmol/L MgCl₂, and 0.8 mmol/L CaCl₂. Experimental hearts received cardioplegia after 15 minutes of preperfusion with each of the following: dimethyl sulfoxide (vehicle), a PKC agonist; acetylcholine (0.55 mmol/L), another PKC agonist; 1,2-s,n-dioctanoylglycerol (DOG, 22 mmol/L) postulated to be further "downstream" in the activation cascade and an irreversible PKC antagonist chelerythrine (CHEL, 0.8 mmol/L). An additional group was pretreated with CHEL, then DOG, to test whether the agonist effect of DOG could be inhibited by this antagonist CHEL. When the 2-hour ischemic period ended, the hearts were reperfused with oxygenated physiologic salt solution at 37°C and the water bath temperature increased to 37°C. Hemodynamic data was recorded every 15 minutes for 45 minutes to compare with baseline data to determine the degree of functional recovery in each heart.

Tissue sampling
To observe changes in tissue nucleotides (adenosine triphosphate [ATP], adenosine monophosphate, adenosine diphosphate, inosine 5'-monophosphate) and nucleosides (adenosine, inosine, hypoxanthine, and xanthine), biopsy of a second paired group of hearts identically handled was performed after they were frozen in liquid N₂ at either baseline, 15 minutes, or the end of reperfusion and then lyophilized. Tissue was processed as previously described by our laboratory [6]. High-performance liquid chromatography was performed with a Waters µBondapak C18 column. The spectrophotometric detector was set at 254 nm for determination of nucleotides and nucleosides and at 210 nm for PCr. Analysis was performed with Waters Maxima 820 software and NEC Power Mate 1. Lactate levels were measured with a lactate analyzer (Yellows Springs Instrument Co, Yellows Springs, OH). Myocardial pH was monitored with a Khuri regional tissue pH monitor (Vascular Technology, Chelmsford, MA).

Determination of PKC translocation
For determination of PKC translocation, hearts were rapidly placed in liquid nitrogen and stored at -70°C. Cytosolic and membrane PKC fractions were prepared as described by Black and colleagues [7]. Briefly, hearts were homogenized in a 20 mmol/L Tris-Cl buffer (pH 7.4) containing 250 mmol/L sucrose; 5 mmol/L EDTA; 0.005% leupeptin; 5 mmol/L benzamidine, and 0.3% ß-mercaptoethanol. The homogenate was centrifuged for 20 minutes at 14,000 g. The supernatant was collected and centrifuged for 105,000 g for 90 minutes. This supernatant contained the cytosolic fraction. The pellet from the initial centrifugation was resuspended and subjected to a centrifugation at 105,000 g for 90 minutes. The pellet was resuspended in buffer and contained the membrane fraction.

The amount of PKC in membrane and cytosolic fractions after induction of PKC-mediated preconditioning was determined by a sandwich-type enzyme-linked immunosorbent assay. Briefly, microtiter plates were coated with a rabbit polyclonal anti-PKC antibody (3.5 µg/mL; Calbiochem, Oakland, CA) specific for the consensus sequence of PKC. Myocyte membrane and cytosolic fractions (50 µg protein/well) were incubated for 90 minutes at 37°C. Wells were incubated with an anti-PKC antibody directed against the PKC isozyme of interest followed by a peroxidase conjugate goat antimouse immunoglobulin G. The chromogen substrate was bephenylenediamine hydrochloride. The optical density was determined at 490 nm using an EL340 automated microplate reader. A standard concentration curve of the PKC isoform was prepared using purified PKC isozyme at concentrations ranging from 0.01 to 100 ng/mL. Studies focused on the PKC isozyme. Antibodies against PKC delta and epsilon are commercially available and have been shown to cross-react with rabbit PKC isozymes. PKC levels are reported as percent of total cellular PKC. Measurement of PKC cytosol and membrane levels required the sacrifice of whole ventricles. Thus, a separate group (n = 6) of rabbits were used to measure these values at baseline and immediately before ischemia for each of the agonists and antagonists investigated.

Heart weight determination
Wet weight of the heart was determined at the conclusion of each experiment after trimming the great vessels and fat and blot drying with nine-layer cotton gauze. The left ventricular wall was weighed, desiccated for 48 hours at 65°C and reweighed. Water content was determined using the formula (1 - dry weight/wet weight) 100%.

Exclusion
Hearts characterized by developed pressures less than 100 mm Hg or greater than 140 mm Hg were not used.

Statistics
Values reported in the text and tables are means ± standard deviation. The Statview 5.01 Program (Abacus Concepts, Inc, Berkeley, CA) was used for statistical analysis. Data were evaluated with analysis of variance (Scheffé’s test). Differences were considered significant when p was less than 0.05.

	Results

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Table 1 summarizes the postischemic metabolic and functional recovery results. There were no significant differences in developed pressure, its first derivative, CF, and MVO₂ among groups during baseline conditions during either phase. We therefore assumed baseline values as 100% to compare the changes in the percentage recovery between the groups during reperfusion. Isovolumic developed pressure was significantly enhanced after ischemia with PKC agonists. This effect was blocked with the PKC antagonist chelerythrine (Fig 1). During preischemia, lactate in coronary effluent was zero in both groups. During ischemia, lactate accumulation occurred during ischemia to a level of 2.22 ± 0.44 mmol/L. There was no difference in lactate levels between groups. During preischemia, myocardial pH was not significantly different between control (7.05 ± 0.10) and DOG hearts (7.06 ± 0.04). In all groups, during ischemia, myocardial pH continued to decrease and at 60, 90, and 120 minutes of ischemia was 6.77 ± 0.24, 6.64 ± 0.23, and 6.56 ± 0.26, respectively. At 5 and 15 minutes of reperfusion, there was no significant pH difference between groups. Total nucleotides (ATP, adenosine diphosphate, adenosine monophosphate, inosine 5'-monophosphate) and nucleosides (adenosine, inosine, hypoxanthine, xanthine) were measured by high-performance liquid chromatography (µmol/L/g). Preischemic ATP values (18 ± µmol/g dry tissue) were equal and by the end of ischemia, myocardial ATP concentration decreased to 3 ± 1 µmol/g dry tissue, without significant differences between groups. Furthermore, 15 minutes after reflow ATP and total nucleotides recovered to equal levels.

View larger version (21K): [in this window] [in a new window]   Fig 1. Percent functional recovery with protein kinase C agonists/antagonists. Recovery of developed pressure (DP) is shown for all the groups and compared as a percentage of preischemic values versus 45 minutes after reflow. Hearts received either protein kinase C agonist acetylcholine (ACH), protein kinase C agonist 1,2-s,n-dioctanoylglycerol (DOG), vehicle dimethyl sulfoxide (DMSO), protein kinase C antagonist chelerythrine (CHEL), or DOG and chelerythrine, with cardioplegia alone used as controls (CTL). (Mean ± standard error of the mean; p < 0.05 for ACH and DOG versus control.)

View larger version (23K): [in this window] [in a new window]   Fig 2. Protein kinase C (PKC) translocation in response to protein kinase C agonists/antagonists, shown as percent of total protein kinase C at membrane. Baseline was after stabilization. Ischemic hearts were subjected to 2 hours of unprotected ischemia. DOG and Chel/DOG hearts received either the protein kinase C agonist 1,2-s,n-dioctanoylglycerol (DOG) or DOG and the protein kinase C antagonist chelerythrine (Chel), administered for 15 minutes and 25 minutes before 120 minutes of cardioplegic induced ischemia.

	Comment

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It has been shown in many studies that PKC mediates preconditioning, including the study by Li and Kloner [11], who showed that the inhibition of PKC abolished the effects of ischemic preconditioning on infarct sizing. These results were confirmed by Bugge and Ytrehus [12], who demonstrated that signal transduction through membrane-bound PKC is essential for preconditioning protection.

Interestingly, differences appear to exist among species in cardiac PKC isoform expression, activation, target proteins, and different cellular functions [13]. It has been shown that PKC alteration of contractility is associated with species-specific cardiac PKC isoforms [14]. In our rabbit model, contractile preconditioning appears to be dependent on activation of the PKC isoform, which was also shown by Meldrum [13] and Armstrong [15] and their colleagues. The mechanism of PKC-induced preconditioning protection is subject to speculation. Many have postulated that it involves an effect of PKC on cytoskeletal contractile proteins. The PKC activation under normal conditions reduces contractile function. Karmazyn and colleagues [16] demonstrated that phorbol ester administration reduced LV contractility in rats. Venema and Kuo [5] demonstrated PKC-mediated phosphorylation of troponin I and C in isolated myocytes, through inhibition of myofibril actomyosin Mg-ATPase. This PKC downregulation of contractile activity during ischemia was also demonstrated by Noland and co-workers [17, 18] who showed PKC phosphorylation of cardiac troponin I and P, resulting in inhibition of Ca⁺²-stimulated ATPase activity and a decrease in actomyosin interactions. Protein kinase C also decreases troponin T binding to the tropomyosin actin complex [18]. Alteration of PKC in response to ischemia may be a signal for contractile quiescence. This inactivation of cytoskeletal contractile proteins may serve as an energy-conserving defense mechanism against ischemia. Teleologically, this contractile quiescence would be an appropriate energy-saving response to ischemia, as more than 90% of the energetics of the heart are normally associated with contractility. However, activation of PKC also results in many other intracellular events within the myocyte, which include altered ion pump regulation [2, 3, 19, 20]. Activation of PKC alters H⁺ and Ca⁺² handling and may change intracellular pH and Ca⁺² levels, which could lead to better ischemic tolerance. In this study, although Ca⁺² was not examined, interstitial myocyte pH, lactate, and high energy phosphates were unchanged between groups. Finally, although PKC activation upregulates myocardial gene expression [2, 3], the beneficial effects of PKC activation in this model, temporally were probably not due to changes in transcription or translation, but more likely due to alteration of PKC-mediated phosphorylation states [21, 22]. Although a increased translocation of the PKC isoform was noted in the present study, the amount of this isoform in the active state and specific subcellular location has yet to be determined. In addition, the possibility exists that the translocation of PKC is not directly involved in mediating the myocardial protection after ischemia but is merely a nonspecific event associated with the use of the PKC antagonists.

In conclusion, these data demonstrate that PKC translocation results in improved postischemic function in this model, presumably not by alteration of energetics or metabolism. Activation of PKC may involve alteration of contractile protein activity, which deserves further investigation [4].

	Acknowledgments

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This work was supported in part by grant G41934 from the American Heart Association of Michigan and an unrestricted grant from the 3M-Sarns Research Foundation.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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